INTERNATIONAL UNIVERSITY

SCHOOL OF BIOTECHNOLOGY

ENZYMES- KINETICS

LE HONG PHU, ASSOC. PROF. PHD

FUNDAMENTAL BIOCHEMISTRY
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 Learning objectives
1. Comprehend how enzyme kinetics
relates to the chemical kinetics
presented in general chemistry
courses.
2. Perceive how kinetic parameters are
experimentally determined.
3. Learn the Michaelis–Menten equation
and the meaning of KM and Vmax.
4. Understand the enzyme kinetics
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.
 Contents
1. ENZYME KINETICS
Michaelis- Menten kinetics
Lineweaver Burk plots
Enzyme inhibition
5. CATALYSIS
Catalytic mechanisms
The role of cofactor in enzyme catalysis
Effects of temperature and pH on enzyme-
catalyzed reactions
Detailed mechanisms of enzyme catalysis
6. ENZYME REGULATION
Covalent modification
Allosteric regulation

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 Activities
 What is scientific method (5 mins)

 Students work online to design an

experiment in science (5 mins)

 Find out how to build up the equation of

the velocity (3 mins)
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 Enzyme kinetics
Kinetics is the study of the rates of
chemical reactions. For any reacting
system, thermodynamics can be used to
predict whether the reaction will
spontaneously occur. The kinetics of the
reaction indicate how fast the reaction
actually goes. Most of the biological
reactions that occur in the cells of living
organisms are greatly sped up by protein
catalysts called enzymes. Recall that
catalysts dramatically increase the rate of
a reaction without affecting the
equilibrium. 5
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 Enzyme kinetics
ELEMENTARY KINETICS

Our first objective for this exercise is to

show how enzyme kinetics relates to the
elementary kinetics you studied in General
Chemistry.
Let’s begin with a simple reaction, a
unimolecular reaction that can be
described as A being converted into B.
A B
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 Enzyme kinetics
Recall that since there is only one reactant, this is a
first order reaction whose rate, or velocity, can be
described as the disappearance of the reactant over
time or the appearance of the product over time. The
reaction rate is represented by the slopes of these
lines and is not constant. For this first-order reaction,
the rate is directly proportional to the concentration
of reactant. Here K is the first-order rate constant for
the reaction. Although the rate constant does not
change, the velocity of the reaction begins to
decrease as the concentration of the reactant
diminishes and the product accumulates. The rate of
the reaction eventually slows to zero as the product
of the initial forward reaction becomes a reactant for
the reverse reaction. Equilibrium is the pointwhere
the forward and reverse reaction rates are equal, and
the overall rate of the reaction is zero. 11
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 Enzyme kinetics
In a simple enzyme-catalyzed reaction, the
reactants are labeled S for substrates and
products are labeled P. This looks like our first
example, a simple first-order reaction in which
substrate is converted to product. Similarly, the
rate of an enzyme-catalyzed reaction is either
the disappearance of the substrate or the
formation of the product over time. As the
reaction proceeds, the reverse reaction begins
to compete with the forward reaction until the
rates are equal, and equilibrium has been
reached. 13
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 Enzyme kinetics
The rate equation for a enzyme-catalyzed
reaction with a single substrate is the
same as the rate equation for the simple
nonenzymatic reaction, but only when the
concentration of substrate is so low that
the enzyme has very little chance to
convert it to product. Under these unique
conditions, it appears that the reaction is
first order with respect to substrate.
However, this special case is much too
limiting and rarely applies in biological and
experimental systems. Consequently,
biochemists must use a different model to
describe the kinetics of biological reactions
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catalyzed by enzymes.
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 Enzyme kinetics
ENZYME KINETICS
The kinetics of an enzyme-catalyzed reaction
can be studied when the concentration of the
enzyme is small compared to the concentration
of the substrate. As long as the substrate is in
large excess over enzyme, altering its
concentration does not change the rate. This is
quite different from the first-order reaction in
the previous example, where the rate
depended on the substrate concentration. Here,
the rate does not depend on the substrate
concentration, and the reaction is said to be
zero order with respect to substrate.
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 Enzyme kinetics
Near the turn of the 20th century, Adrian Brown
first observed this conflicting behavior of
enzyme-catalyzed reactions. It became apparent
to him that the substrate must form a complex
with the enzyme. This observation marked the
beginning of the study of enzyme kinetics. Brown
correctly deduced that a zero-order reaction with
respect to substrate occurred when the enzyme
was saturated with substrate. Essentially, the
limited number of active sites put a ceiling on
how fast the reaction could proceed. Because the
enzyme concentration is rate limiting, the rate is
independent of substrate concentration. The
zero-order rate constant is referred to as the
maximal velocity, or Vmax.
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 Enzyme kinetics
To account for this kinetic behavior caused by
the substrate’s interaction with enzyme, the
model for an enzyme-catalyzed reaction must
include an enzyme-substrate complex, or ES
complex. The reaction scheme is shown here. The
rate constants k1 and k–1 are the forward and
reverse rate constants for the formation of the ES
complex, and k2 is the rate at which the ES
complex converts the bound substrate into
product. Notice that in order to simplify this
model, we assume that the product formation
step is irreversible (that is, there is no k–2).

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 Enzyme kinetics
Catalysts are generally not
considered reactants because they
facilitate reactions without being
altered. However, in an enzyme-
catalyzed reaction, the enzyme does
participate as a “pseudo-reactant”
because it must bind and form a
complex with substrate, and the
availability of its active sites affects
the overall rate of the reaction. 25
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 Enzyme kinetics
MICHAELIS–MENTEN EQUATION
We have seen that for an enzyme-
catalyzed reaction, we can write a
simple rate equation only when the
concentration of substrate is
saturating or when the concentration
of enzyme is saturating. In between
these two extremes, we need to use
a slightly more complex rate
equation called the Michaelis–Menten
equation. 28
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 Enzyme kinetics
The Michaelis–Menten equation is limited
to the initial rate v0 so that we can ignore
the possibility of the product accumulating
and undergoing the reverse reaction. The
Michaelis–Menten equation and its
hyperbolic graph describe how the
reaction rate varies with substrate
concentration for a one-substrate
reaction. It is important to note that
despite the similarities in shape, this
graph is NOT a graph of the concentration
of product forming over time.

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 Enzyme kinetics
This graph shows the reaction
progress of an enzyme-catalyzed
reaction. The Michaelis–Menten
model makes a steady-state
assumption, meaning that the
concentration of the ES complex
remains constant. This assumption is
valid only when the concentration of
substrate is much greater than the
total enzyme concentration.
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 Enzyme kinetics
KINETIC PARAMETERS
Vmax is the maximal velocity that can be
achieved by an enzyme under the special
case of saturating substrate concentration.
KM is a “lumped” rate constant
incorporating all the rate constants for ES
and P formation: k1, k–1, and k2. It is
equal to the substrate concentration that
gives 1/2-saturation of the enzyme. It is
therefore also equal to the substrate
concentration at one-half the Vmax.
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 Enzyme kinetics
For a reaction that follows the Michaelis–Menten
kinetic model, the KM is inversely related to the
proportion of total enzyme (Et) which is in complex
with substrate. In other words, the higher the KM, the
lower the fraction of enzyme with a bound substrate.
Although loosely related, the KM is not the same as
the enzyme’s affinity for substrate, for it also takes in
to account the decomposition of the ES complex to
product. The most useful definition for KM is that it is
a constant that describes the dependence of v0 on S,
and dictates the steepness of the shape of the
Michaelis–Menten curve.
Experiment with the sliders to change Vmax and KM.
Note how the shape of the line changes with KM.
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 Enzyme kinetics
A KINETIC EXPERIMENT

To help you understand the

hyperbolic relationship between the
substrate concentration and the rate
of an enzyme-catalyzed reaction
which is described by the Michaelis–
Menten equation, we will now
demonstrate a kinetic experiment.
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 Enzyme kinetics
To experimentally determine the relationship
between substrate concentration and initial
velocity, a biochemist sets up a series of test
tubes for the reaction. Each tube contains a
constant amount of enzyme. But the amount of
the substrate placed in each tube varies. For
easy analysis, the biochemist often chooses a
substrate that yields a product that has a
different color or different fluorescence. In
order to measure the initial velocity, the
reaction is only run for a short length of time.
This is done so that only a small percentage of
substrate is converted into product, and
therefore there will be no kinetic contribution
from the reverse reaction of product formation,
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and k–2 can still be ignored.
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 Enzyme kinetics
The amount of product formed over
time is monitored. The amount of
product formed divided by the brief
time of incubation is the slope of the
early linear phase of the reaction.
This rate of product formation is the
initial velocity of the reaction. Note
that the tubes containing the most
substrate yield the highest initial
velocities. 47
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 Enzyme kinetics
A plot of initial velocity versus substrate
concentration yields a hyperbolic curve
consistent with the Michaelis–Menten model of
enzyme activity. The important kinetic
parameters Vmax and KM can be estimated
from this graph. The Vmax is the point where
the enzyme is fully saturated and cannot
achieve a higher initial velocity Vmax and is
therefore the upper limit for v0. The KM is the
substrate concentration that corresponds to
1/2 of the Vmax. More precise values for
Vmax and KM can be obtained by using a
curve-fitting program. Remember that the
Michaelis–Menten equation is a mathematical
expression for a hyperbola.
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 Enzyme kinetics
VIRTUAL ENZYME KINETICS

Now imagine that you are a biochemist

attempting to characterize the kinetic behavior of
a new mutant of the motor protein kinesin You
will need to set up reaction tubes and collect data
to determine the values for Vmax and KM. You
will be monitoring the ATPase activity of the
kinesin by quantifying the conversion of
radiolabeled ATP to ADP.

Plan your experiment by indicating the proper

order of the steps.

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 Enzyme kinetics
First, prepare six 1 milliliter reaction
samples. Use the micropipettor to
add samples drop-wise to the tubes.
Be careful: sometimes the order of
addition matters. Be sure to leave
room in the tube for substrate, and
do not add substrate to any tube
until all samples are ready. When all
the samples are prepared, add
substrate to each tube and start the
timer. 56
 Enzyme kinetics
Now that you are ready to start the
reactions, add substrate and start the
timer. After the desired incubation time,
quench the reactions with detergent to
inactivate the enzyme.
You now need to determine the amount
of product formed in each reaction tube.
You will use thin layer chromatography to
separate the ADP product from the
leftover ATP substrate and quantify the
radiolabeled ADP by phosphoimaging and
densitometry. The values obtained are
now added to the table.
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 Enzyme kinetics
From the experimental data, calculate the initial velocities
and enter the values into the last row of the table.
Now use your data to construct a Michaelis–Menten plot.
First, select the proper axis labels, then add your x and y
data to plot a curve. While you could roughly estimate
Vmax and KM by eye or achieve more precise values by
fitting the data computationally, it is instructive to do the
curve fitting by hand. Use the sliders for Vmax and KM to
create a curve that is computed from the Michaelis–Menten
equation and that fits your data. Note how changing each
kinetic parameter affects the shape of the curve.
Manipulate the parameters until you are satisfied with the
fit, and then press "Accept fit."

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 Enzyme kinetics
From your data you have obtained
values for Vmax and KM. You can now
use these kinetic parameters to compare
the ATPase activities of your kinesin
mutant and wild-type kinesin, in order to
determine the effect of the mutation. This
information, coupled with the location of
the mutation in the three-dimensional
structure of kinesin, could lead to new
insights into the mechanism of this motor
protein.
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 Enzyme kinetics
LINEWEAVER-BURK PLOTS

While computers can easily determine kinetic

parameters from a hyperbolic Michaelis–Menten
plot, these graphs are unwieldy for visually
estimating values for Vmax and KM. For this
purpose, the Michaelis–Menten equation can be
rearranged to a linear equation with the form y =
mx + b. The resulting plot is called a Lineweaver-
Burk or double-reciprocal plot. KM and Vmax can
be readily obtained from the x and y intercepts of
a Lineweaver-Burk plot. The y-intercept is
1/Vmax and the x-intercept is –1/KM.Try varying
the Vmax and KM on the following graphs.
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 Enzyme kinetics
CONCLUSION

Chemical kinetics and enzyme kinetics are

related, but because of the complex of enzyme
with bound substrate, enzyme kinetics only
correlates well with chemical kinetics in the
extreme cases with very limited substrate or vast
excess of substrate.

Michaelis–Menten enzyme kinetics describes the

behavior of enzymes over a wide range of
substrate concentrations. Two important kinetic
parameters are Vmax and KM.

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 Enzyme kinetics
Vmax is the maximal velocity of an enzyme-
catalyzed reaction at saturating substrate
concentration. KM is loosely related to
substrate affinity, but a more correct
description is that it determines the shape of
the Michaelis–Menten hyperbolic curve. Kinetic
experiments can quantify how the rate of an
enzyme-catalyzed reaction changes with the
concentration of substrate. Data obtained can
be plotted and fit to the Michaelis–Menten
equation or rearranged into a double-
reciprocal plot. From these graphs, the kinetic
parameters KM andVmax can be obtained. 63
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 Enzyme inhibitor
INTRODUCTION
Almost all therapeutic drugs are enzyme
inhibitors, from old medicine box
standards such as aspirin and penicillin to
the newest compounds used to treat HIV
infection. Understandably, enzyme
kinetics plays an outstanding role in this
effort to produce effective therapeutics,
for kinetic studies can quantify the degree
that inhibitors inactivate or slow down the
targeted enzyme’s catalytic rate and
describe its potential efficacy as a drug. 67
 Enzyme inhibitor
In addition, many naturally produced
toxic substances and chemical warfare
agents are also enzyme inhibitors. For
example, a peptide found in snake venom
became a lead compound in the
development of a class of hypertension
drugs that inhibit angiotensin-converting
enzyme (ACE inhibitors). The nerve gas
sarin is a potent inhibitor of an enzyme
important for synaptic transmission
innerve tissue. 68
 Enzyme inhibitor
Enzyme inhibitors alter the activity
of an enzyme by decreasing the
enzyme’s ability to bind substrate
Lowering its catalytic turnover, or
both.

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 Enzyme inhibitor
OVERVIEW: REVERSIBLE VS.
IRREVERSIBLE INHIBITORS

There are two main classes of enzyme

inhibitors, reversible and irreversible, that
are differentiated by the magnitude of
their affinity for enzyme. Reversible
enzyme inhibitors bind and dissociate with
their enzyme in a equilibrium process.
Irreversible inhibitors bind tightly to an
enzyme to form an essentially permanent
complex.
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 Enzyme inhibitor
IRREVERSIBLE INHIBITORS

Because they form such a tight complex with

the enzyme that they permanently remove its
catalytic activity, irreversible inhibitors are also
termed inactivators. For example, kinetic
studies on the male pattern baldness drug
finasteride (Propecia®) reveal it to be an
essentially irreversible inhibitor of the 5-α-
reductase. This enzyme catalyzes one step in
the conversion of testosterone to
dihydrotestosterone, which is a hormone
implicated in hair loss. 71
 Enzyme inhibitor
Some irreversible inhibitors even
become covalently bound to amino
acids in the active site of the enzyme
as they are brought through the
early chemical steps of catalysis.
Aspirin is such an example.

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 Enzyme inhibitor
REVERSIBLE INHIBITORS
Reversible inhibitors can be classified as
competitive, mixed, or noncompetitive
inhibitors. If the detailed mechanism of
inhibition is known, then the classification
can be made by identifying where on the
enzyme the inhibitor binds, or the order
with which it binds, relative to substrate.
Alternatively, a determination of simple
kinetic parameters can generally be used
to classify the inhibitor.
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 Enzyme inhibitor
COMPETITIVE INHIBITION

Competitive inhibitors compete with

substrate for an enzyme’s active site,
lowering the enzyme’s likelihood of
binding substrate and slowing the
observed reaction velocity.

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 Enzyme inhibitor
Competitive kinetics
Kinetic studies can be used to determine
the type and potency of inhibition for an
unknown inhibitor. Typical steady-state
kinetic experiments can be performed
where reaction velocity is measured in the
presence of varying concentrations of
substrate. If inhibitor is then added, and
the data shows an increase in KM, yet the
Vmax is unaffected, this is the signature
of a competitive inhibitor.
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 Enzyme inhibitor
MIXED INHIBITION
A mixed inhibitor binds to a site on the
enzyme and interferes with both apparent
substrate affinity and catalytic turnover,
thus affecting both the observed KM and
kcat for the enzyme-catalyzed reaction.
Mixed inhibitors do not bind directly in
the active site, and therefore do not block
substrate binding, but instead bind at sites
that can be proximal or distal from the
active site.
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 Enzyme inhibitor
Mixed inhibitors can therefore bind to
free enzyme prior to substrate,
distorting the active site to a
nonoptimal conformation for
catalysis. The inhibitor-distorted
active site has trouble converting the
substrate to product before it
dissociates, resulting in a lowered
apparent substrate binding affinity.
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 Enzyme inhibitor
Mixed inhibitors distort the active site,
interfering with the chemistry performed
by the enzyme. Therefore the enzymatic
turnover rate is slowed.
Mixed inhibitors can also bind after
substrate to the enzyme-substrate
complex. Similar to before, the inhibitor
binding to the ES complex distorts the
active site and its bound substrate to a
nonoptimal conformation, so that less ES
complex productively form product before
the substrate dissociates.
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 Enzyme inhibitor
Binding of the mixed inhibitor to the
ES complex also interferes with the
subsequent chemistry step.

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 Enzyme inhibitor
Mixed kinetics

Steady-state experiments performed

in the presence of a mixed inhibitor
demontrate an increase in KM, and a
decrease in Vmax. Remember, Vmax
is simply kcat multiplied by the total
enzyme concentration. Therefore, a
decrease in kcat is a decrease in
Vmax.
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 Enzyme inhibitor
NONCOMPETITIVE INHIBITION

Noncompetitive inhibition is a special

case of mixed inhibition where the affinity
of inhibitor for E and ES is the same.
Noncompetitive kinetics Steady-state
experiments performed in the presence of
a noncompetitive inhibitor demonstrate a
decrease in Vmax, yet KM is unaffected.

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 5. CATALYSIS

Catalytic mechanisms

 The role of cofactor in enzyme

catalysis
 Effects of temperature and pH on
enzyme-catalyzed reactions
 Detailed mechanisms of enzyme
catalysis
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 6. ENZYME REGULATION
Covalent modification
Allosteric regulation

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ANY QUESTIONS

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Thank you for your kindly listening

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